CHOLESTEROL METABOLISM AND MITOCHONDRIAL CYTOCHROME P450

在所有类固醇生成细胞中，胆固醇的侧链被 细胞色素P450scc决定总类固醇生成率和 荷尔蒙调节的。在肾上腺束状细胞和肾小球细胞中， 过程由不同的信令路径控制(分别， ACTH/cAMP和血管紧张素/钙)。胆固醇向细胞色素P450scc转移 通过荷尔蒙的刺激将胆固醇转移到 线粒体和胆固醇在线粒体之间的转移 膜。随之而来的是细胞内游离胆固醇的增加 通过细胞骨架的运动，可能是由甾醇载体促进的 蛋白质2。其他因子(类固醇生成激活素(SAP)， Endozepine，5-HPETE]与线粒体内有关 胆固醇转移。这可能需要专门的联系人站点，由 外膜外周苯二氮卓类受体(由 Endozepine)和内膜，受激素刺激30 kDa 磷蛋白。我们将使用最近开发的渗透性肾上腺 研究激活胆固醇机制的细胞准备 调职。我们还将纯化30 kDa线粒体蛋白(P30)。 以进一步确定其在肾上腺线粒体中的作用。在这两个地方 束状细胞和肾小球细胞，ACTH和血管紧张素， 刺激相同多种形式的P30的合成，表明 类似的激活过程。大鼠肾上腺束状细胞 用链球菌溶血素O渗透(&gt；90%台盼蓝摄取)，显示双倍 完整细胞的活性，保留了50倍刺激产生的 ACTH在通透性之前，在通透性之后被刺激 通过cAMP和Ca~(++)。我们将对这些进行优化，然后对其进行描述 与完整的肾上腺细胞有关的准备。他们将习惯于 分析束状藻和白纹伊蚊胆固醇代谢的激活 在胆固醇转移方面，肾小球细胞。超微结构 与通透性和激素激活相关的变化将 接受检查，特别是细胞骨架和胆固醇 分发。我们将确定在多大程度上完好无损的过程 细胞被渗透化的细胞保留；值得注意的是，激活 胆固醇酯酶、P30和P30的合成及磷酸化 胆固醇代谢。渗透细胞将被用来测试 特定蛋白质和介体的参与，或者通过直接摄取 或者通过添加特定的抗体。线粒体被激活 通透性细胞中的潜在介体(Ca++、Endozepine、5-HPETE) 与对分离的线粒体的影响相比，我们将继续 努力分析多种形式的P30的细胞合成。我们 目的纯化牛肾上腺P30(或使用在别处建立的序列) 为了产生将用于定位P30的特定抗体 线粒体中，并确定相关蛋白质。这项工作将 建立一种新的分析类固醇合成的实验方法 来自所有类固醇生成器官的细胞。

In all steroidogenic cells, the side-chain cleavage of cholesterol by cytochrome P450scc determines the rate of total steroid production and is hormonally regulated. In adrenal fasciculata and glomerulosa cells, this process is controlled by distinct signalling pathways (respectively, ACTH/cAMP and angiotensin/Ca++). Cholesterol moves to cytochrome P450scc through hormonal stimulation of both movement of cholesterol to the mitochondria and then cholesterol transfer between mitochondrial membranes. An increase in free cytosolic cholesterol is followed by movement through the cytoskeleton, possibly facilitated by sterol carrier protein-2. Other factors (steroidogenesis activator peptide (SAP), endozepine, 5-HPETE] have been implicated in intramitochondrial cholesterol transfer. This may require specialized contact sites formed by outer membrane peripheral benzodiazepine receptors (activated by endozepine) and an inner membrane, hormonally stimulated 30 kDa phosphoprotein. We will use a recently developed permeabilized adrenal cell preparation to address the mechanism of activated cholesterol transfer. We will also purify the 30 kDa mitochondrial protein (p30) in order to further define its role in adrenal mitochondria. In both fasciculata and glomerulosa cells, ACTH and angiotensin, respectively, stimulate synthesis of the same multiple forms of p30, suggesting a similar activation process. Rat adrenal fasciculata cells which have been permeabilized (>90% trypan blue uptake) with streptolysin O, show double the activity of intact cells, retain the 50-fold stimulation generated by ACTH prior to permeabilization, and are stimulated after permeabilization by cAMP and Ca++. We will optimize and then characterize these preparations in relation to intact adrenal cells. They will be used to analyze the activation of cholesterol metabolism in fasciculata and glomerulosa cells in terms of cholesterol transfer. Ultrastructural changes associated with both permeabilization and hormonal activation will be examined, with particular reference to cytoskeleton and cholesterol distribution. We will determine to what extent the processes of intact cells are retained by the permeabilized cells; notably, activation of cholesterol esterases, synthesis, and phosphorylation of p30 and cholesterol metabolism. Permeabilized cells will be used to test the participation of specific proteins and mediators, either by direct uptake or by addition of specific antibodies. Mitochondrial activation by potential mediators (Ca++, endozepine, 5-HPETE) in permeabilized cells will be,compared to effects on isolated mitochondria: We will continue efforts to analyze the cellular synthesis of the multiple forms of p30. We aim to purify bovine adrenal p30 (or use sequences established elsewhere) in order to generate specific antibodies that will be used to localize p30 in the mitochondria, and to determine associated proteins. This work will develop a new experimental approach for analysis of steroidogenesis in cells from all steroidogenic organs.